When producing recombinant proteins at industrial scale, one must isolate clones producing high amounts of recombinant proteins.
Introducing heterologous genes into animal host cells and screening for expression of the added genes is a lengthy and complicated process. The process involves the transfection and the selection of clones with stable long-term expression, and the screening for high expression rates of the recombinant protein.
When generating clones expressing a recombinant protein from expression vectors, host cells are usually transfected with a DNA vector encoding both the protein of interest and the selection marker on the same vector. Such an expression vector thus comprises a selectable marker allowing the selection of clones in which the expression vector is present. Such a selectable marker may also lead to a co-amplification taking place, thereby allowing the isolation of high-producer clones.
Several such selectable markers are known in the art, including e.g. G418, hygromycin, puromycin, zeomycin, dihydrofolate reductase (DHFR), glutamine synthetase (GS) and hypoxanthine-guanine phosphoribosyltransferase (HPRT). In particular, GS is widely used as a selectable marker in the field of industrial recombinant protein production in eukaryotic cells.
More specifically, WO 87/04462 describes the use of glutamine synthetase (GS) as a selectable marker. The examples teach an expression vector comprising, as a selectable marker, the sequence coding for a GS of Chinese hamster origin. It is further shown that such an expression vector allows production of a recombinant protein upon transfection of the expression vector into CHO cells, the recombinant protein being tPA.
Even though the above CHO expression system based on the use of GS as a selectable marker was described as early as in the 80'ies, it remains a standard in the art still today. In particular, no significant improvement to the original GS selectable marker has been published.
Indeed, the Korean patent KR10-0267720 discloses the use of human GS as a selectable marker. However, the exact sequence of the human GS used is not disclosed. Moreover, it is also indicated the technical effect (high yield) is only linked both with the human GS and with the specific SV40 promoter that is used (i.e. an SV40 promoter that lacks positions 128 to 270).
There is thus a need in the art for additional and/or improved expression systems allowing the isolation of a high number of clones expressing the recombinant protein for which production is desired, at least some of these clones exhibiting high expression rates of the recombinant protein.